1. Area of the Art
The invention relates generally to solid supports with immobilized biopolymers and specifically to solid supports with immobilized unmodified biopolymers and methods of immobilizing unmodified biopolymers to solid supports.
2. Description of the Prior Art
Biopolymer synthesis and biopolymer analysis often require the attachment of biopolymers to solid supports. For example, organic and inorganic materials have been utilized for the solid phase synthesis of peptides, oligonucleotides and small organic molecules. The synthesis involves the stepwise addition of activated monomers such as amino acid derivatives or nucleotide derivatives to a growing oligomeric chain attached at one end to a solid support. At the completion of the synthesis, the newly synthesized biopolymers may be cleaved from the solid support and subsequently utilized in biochemical research or diagnostic applications or, alternatively, be utilized without cleaving the biopolymers from the solid support.
For biopolymer analysis, biopolymers may be attached to a solid support in several ways. In blotting techniques, native biopolymers are first captured onto a membrane and subsequently immobilized on the membrane by heat, radiation or chemical techniques. The immobilized biopolymers are then available for subsequent analyses, such as those associated with southern blotting applications and reverse hybridization analytical techniques.
Additionally, presynthesized or natural oligonucleotides have been immobilized by covalently attaching activated oligonucleotides to the solid support. Current methodology for the covvalent attachment of nucleic acids to solid supports (substrates) involves modification of the DNA (or RNA). For example, oligonucleotides are usually derivatized to a 5'-amino terminus, making the DNA more reactive for covalent attachment to an activated surface. Other methods of attachment have employed reactions with terminal phosphate groups or Sulfhydral groups with surface carbodiimide or other activation chemistries (see Lund et al, Nucleic Acid Res. 16:10861-80, 1988, Bischoffet al, Analyt. Biochem. 164: 336-344, 1987).
It is generally understood that reactive groups present within native polynucleotides are weak and therefore make for inefficient attachment. In addition, when native polynucleotides are exposed to highly reactive surface groups, excessive crosslinking may occur. This crosslink may prevent the attached nucleic acid from fully participating in hybridization. These conditions are most noticeable for short fragments of double-stranded DNA or oligonucleotides. Thus, oligonucleotides often have to be modified, for example, derivatized to a 5'-amino terminus, for an effective attachment. The 5' amino-linker allows selective binding of the amino-containing DNA to silylated slides through a Schiff's base reaction with aldehyde groups on the chip surface. The selectivity of amino-modified versus natural, unmodified DNA is about 10:1 for cDNAs and about 10,100:1 for single-stranded 15-mers. DNA molecules of intermediate lengths exhibit intermediate discrimination ratios. In addition, the 5' end attachment of the DNA to the chip via the amino group permits steric accessibility of the bound molecules during the hybridization reaction. Therefore, post-modification has been perceived as obligatory for attachment of, e.g., oligonucleotide probes for creation of arrays. Such post-modification processes require additional time-consuming steps at substantial costs.
Therefore, it is desirable to develop a more effective method for attaching biopolymers, particularly unmodified biopolymers to a solid support. It is particularly desirable to develop a method to directly attach unmodified biopolymers, such as polynucleotides, to a solid support.